Robert C. Rizzo, Ph.D. - US grants

DESCRIPTION (provided by applicant): The molecular mechanisms of resistance to viral entry fusion inhibitors targeting HIVgp41 are not well-understood. Fundamental gaps in knowledge of the energetic and structural interactions which drive binding hamper the long-term goal of development of new drugs with improved resistance profiles. The overall objective of this application is to (1) develop computational structural models to quantify binding for known gp41 fusion inhibitors (both peptides and small molecules), (2) characterize origins of resistance profiles to current inhibitors, and (3) discover new small molecule drug-leads. Based on strong preliminary results, the central hypothesis is that specific interactions within a conserved hydrophobic pocket on gp41, not exploited by the only currently available anti-fusion drug (peptide inhibitor T20), confer improved resistance profiles to next-generation peptide inhibitors and drive binding for small molecule inhibitors. The rationale for the proposed research is that robust computational models allow drug binding to be fully characterized at the atomic level, and this will enable development of HIV drugs with favorable resistance profiles. Thus, the work proposed is directly relevant to the NIH plan for basic and applied research towards discovery and development of novel agents and therapeutic strategies directed against viral factors involved in HIV replication and persistence. The work employs all-atom computer simulations (molecular dynamics and docking), in conjunction with detailed energetic and structural analysis, to test the central hypothesis and accomplish the goals set forth in each specific aim. Aim #1 will determine the molecular basis of resistance to current peptide fusion inhibitors of gp41 to test the hypothesis that binding affinity for T20 is driven primarily by interactions with mutation-prone regions along the binding interface. Aim #2 will characterize the mechanism of action for reported small molecule inhibitors of gp41 which we postulate are due to specific energetic and structural interactions modulated within the conserved pocket. Aim #3 will identify new small organic molecules, which bind specifically to the gp41 pocket, using virtual-high-throughput-screening in conjunction with experimental validation. Active compounds will be characterized structurally using NMR and X-ray crystallography and developed further. The proposal's contributions are significant because results from detailed binding models and computer simulations will allow the molecular basis of recognition to be delineated, which will enable development of improved fusion inhibitors that maintain activity against clinically relevant HIV escape mutations. PUBLIC HEALTH RELEVANCE: Results from the proposed research will be used to uncover the atomic-level structural and energetic determinates which describe binding of membrane fusion inhibitors with the viral entry protein gp41 which mediates HIV infection. The proposal seeks to understand the origins of resistance to gp41 inhibitors, and develop new compounds with improved resistance profiles, thus the finding are expected to be of direct relevance to public health.

PROJECT SUMMARY/ABSTRACT The mosquito-borne Zika virus (ZIKV), which the WHO has declared a global emergency, is spreading explosively in the Americas and as many as 4 million people could become infected by the end of 2016. From a global health perspective, the severe microcephaly and associated birth defects arising from infection make development of effective therapeutic agents for ZIKV paramount. The goal of this proposal, written in response to the recent call titled Rapid Assessment of Zika Virus (ZIKV) Complications (R21) (PAR-16-106) is to identify small molecule drug-leads that inhibit ZIKV entry and replication. Our overall objective is to develop inhibitors capable of blocking key conformational changes required for membrane fusion that involve the ZIKV envelope glycoprotein (E). We hypothesize that structure-based computational screening using knowledge of viral interfaces followed by experimental characterization will be more successful than random screening. The rationale for proposing the work, is the ability of our team to rapidly leverage prior experience gained targeting analogous events in HIV and Ebola and the ability to construct robust structural models for ZIKV screening based on the high homology of dengue virus glycoprotein E (74% similarity, 54% identify) for which abundant crystallographic structural information is available. The project is arranged as two Specific Aims according to the expertise of each PI's lab: (Aim #1) Identify and exploit targetable events in ZIKV glycoprotein E. Under this aim we will computationally target favorable protein interfaces to disrupt membrane fusion using high- throughput-virtual screening of ca. 5 million commercially available compounds. Three distinct E protein sites will be targeted, based on ZIKV models derived from related protein structures of dengue virus reported by Harrison and coworkers, include the previously described ?-OG pocket (pre-fusion state) and two pockets we have identified based on analysis of a recently reported late-stage fusion intermediate. Screening will employ atomic-level footprints to identify the most promising top-scoring compounds (300-400) for experimental characterization. (Aim #2) Characterize small molecule probes identified computationally by quantifying the disruption of viral entry. Top-scoring compounds (300-400) predicted to arrest entry by disrupting membrane fusion will be tested in Vero cells and human fibroblasts (HFF-1). Initial screens will be performed in a high- throughput non-replicating pseudotyped virus system (ZIKV M/E protein in an HIV particle with a luciferase reporter). Positive hits will be tested with live ZIKV infectivity assays (plaque assay and endpoint dilution assay). Experimental testing will allow prioritization of the molecules identified by virtual screening. Broader impacts of the work include increased understanding of how to target related flaviviruses including dengue, yellow fever, tick-borne encephalitis, and West Nile.

PROJECT ABSTRACT Tsg101 is a component of cellular ESCRT-I (endocytic sorting complex required for transport-I) and plays a well-established role in budding of HIV-1 and other pathogens from infected cells. In the case of HIV-1, the interaction occurs through a Pro-Thr-Ala-Pro (PTAP) motif in the HIV-1 structural precursor polyprotein, Gag. Ubiquitin (Ub) is also an important participant in Gag recognition. Translating these critical virus-host protein-protein interactions into useful therapeutic targets would have broad impact on anti-viral drug development strategy. Initially, random screening of a library of small molecules currently in use or in development as drugs for other indications was used to identify small molecules capable of blocking the interaction of Gag with Tsg101 without affecting cellular Tsg101 functions. We solved the NMR structure of Tsg101 in complex with a promising lead compound and now propose to improve the lead and discover additional small molecule inhibitors by conducting structure-activity relationship analyses (SAR) and computer-aided structure-based design. Innovative computational tools developed by our team for targeting protein-protein interfaces will be used to exploit the unique energetic and structural information inherent to molecular footprints (interaction maps) made by the initial lead, as well as PTAP, and Ub proteins in their respective binding interfaces within the Ub E2 variant (UEV) domain on Tsg101. The goal is identify small molecules compatible with the interfaces through virtual screening of large commercially available ligand libraries. It is anticipated that targeting Tsg101 rather than virus-encoded gene products as is currently done will circumvent several challenging problems associated with management of HIV infections, including resistant virus emergence, and potentially provide even wider impact through broad-spectrum application to other human pathogens requiring Tsg101 for replication. The specific goals are: (Aim 1) To conduct experimental SAR analyses and validation strategies on a recently identified targetable region in the Tsg101 UEV domain; (Aim 2) To conduct computational SAR and validation strategies that permit identification and refinement of new inhibitors using validated computational tools based on molecular footprints of PTAP, Ub, and the lead in their respective UEV binding pockets.

PROJECT SUMMARY/ABSTRACT The central objectives of this research proposal are development and validation of methodologies to algorithmically encode underlying physical observables to improve design of small organic molecules for a biological target and their application to real world systems. Computational modeling at the atomic-level empowers understanding of the factors that drive molecular recognition and enables testable predictions that can be confirmed by experimentalists. Grounded in strong results and data, we hypothesize that major gaps in the field (i.e. pose accuracy, enrichment, protein flexibility, specificity, site complementarity, ease of use) can be bridged through forward-thinking design of tools that improve sampling, scoring, and searching. A major undertaking is development of a new platform for de novo design which will enable from-scratch construction of novel molecules, which removes the limitation of only considering those that are preconceived. This will enable design of compounds highly optimized and specifically tailored to the protein of interest. Our approach employs construction of molecules starting from user customizable libraries of building block fragments using algorithms we developed and implemented into the program DOCK6. New advances will be made available to the research community through public releases along with validation databases and user- friendly online tutorials. Without inventive approaches to ligand discovery, there is a high likelihood that certain areas of chemical space may not be adequately sampled by standard screening methods which provides the rational. Our expected outcomes are ensembles containing highly specific and optimized ligands. The proposal is framed around 4 fundamental questions: (Q1) What underlying physical principles that drive molecular recognition (binding, selectivity, resistance) can be captured at the atomic-level and used to design improved software and simulation protocols for accurate prediction of geometry and energy? (Q2) Can ligand growth be propelled to highly specific regions of chemical space through from-scratch assembly of small organic fragments (de novo design) using molecular mimicry principles to direct the growing ensemble as it evolves? (Q3) Which sampling, scoring, and searching methods are most effective for identification and design of verified-active compounds and can more effective practices be developed to maximize overall success in collaboration with experimentalist? (Q4) Can docking and de novo design software and protocols be designed to be more user friendly while not sacrificing accuracy or power? We will collaborate with a network of experienced experimental labs and employ our new tools to make predictions. We will identify small molecule probes and inhibitors to answer basic research questions and provide mechanistic understanding for biological systems of relevance to human health including: fatty acid binding protein, nSMase2, neutral ceramidase, HIVgp41, GP2, glycoprotein-E, ErbB-family mutants (EGFR, HER2), candid albicans Glx3/Hsp31, and human Tsg101, among others. Experimental outcomes in turn will inform our further method development efforts.

Project Summary Despite advances in anti-androgen and taxane-based therapies, prostate cancer (PC) often becomes castration-resistant, metastatic, and incurable. Consequently, there is an urgent need to develop novel interventions to treat metastatic PC. Lipid signaling and metabolism are major drivers of PC metastasis and present ideal targets for therapeutic intervention. However, therapeutic exploitation of lipid signaling systems is hampered by the existence of multiple lipid metabolizing enzymes and nuclear receptors, which would necessitate targeting these systems in parallel. Fatty acid binding protein 5 (FABP5) is an intracellular carrier that shuttles bioactive lipids to nuclear receptors, thereby activating gene transcription programs that enhance tumor growth and metastasis. FABP5 is not expressed in the normal prostate but becomes highly upregulated in advanced metastatic PC. Our group has obtained preliminary data demonstrating that FABP5 is indispensable for the delivery of pro-tumorigenic lipids produced by multiple cytosolic to nuclear receptors to promote PC metastasis. This positions FABP5 as an essential node in a PC lipid signaling network and an attractive target for the development of therapeutics to treat metastatic PC. Despite the considerable promise of FABP5 inhibitors as potential PC therapeutics, potent and selective inhibitors have yet to emerge. The major goal of this proposal is to develop and optimize novel potent and selective FABP5 inhibitors. The proposed multidisciplinary project will be carried out by a highly qualified team with expertise in computer-aided drug design, medicinal chemistry, and PC biology. Aim 1 will leverage structure-based drug design and iterative chemical synthesis approaches to identify and optimize FABP5 inhibitors for potency and selectivity. Aim 2 will employ a robust in vitro inhibitor testing platform including assessments of inhibitor potency, efficacy, selectivity, stability, and cytotoxicity in PC cell-lines and non-transformed cells. Aim 3 will assess the efficacy of candidate inhibitors in mouse models of PC, including a novel genetically engineered mouse model of androgen-dependent and castration-resistant PC. We will also assess the efficacy of FABP5 inhibitors when used as monotherapies and in combination with FDA approved therapeutics. Successful completion of the proposed studies will lead to the development of optimized FABP5 inhibitor scaffolds that can be advanced to late stage IND-enabling studies and eventual clinical deployment.